Innovations in and improvement of bio-downstream processing, which is responsible for about 50-80% of recombinant proteins and other biomolecules, play a very important role in increasing the yield and reducing the cost of biopharmaceutical production. Biomolecule isolation and purification from a fermentation broth usually involve centrifugation, filtration, adsorption, and chromatography steps. Each step contributes to the product cost and product loss.
Bioproduct recovery from fermentation broths is complicated by the large number of dissolved substances and suspended particles present in the broth. Most bioseparation processes involve the following steps: removal of insolubles by either filtration or centrifugation, isolation of products using either adsorption or solvent extraction, purification (via chromatography and precipitation) and polishing via crystallization, or spray drying and lyophilization. For intracellular products, cell disruption is needed also to release the product before the removal of insolubles. See: Belter, P. A., Cussler, E. L. and Hu, W. Bioseparations: Downstream Processing for Biotechnology, John Wiley & Sons, Inc. (1988). Although the series of separation steps can usually accomplish the product recovery, reduction in the overall number of separation operations is desirable because of the low product yields associated with some steps. The development of techniques that reduce the overall number of steps are gaining popularity due to their reduced cost, increased yield and productivity along with the reduction in the complexity of the downstream processing flowsheets. See: Agrawal, A. and Burns, M. A., Biotechnology and Bioengineering, 52, 539 (1996); Chang, Y. K. and Chase, H. A., Biotechnology and Bioengineering, 49, 204 (1996); Freeman, A., Woodley, J. M. and Lilly, M. D., Bio/Technology, 11, 1007 (1993); and Molinari, R., Torres, J. L., Michaels, A. S., Kilpatrick, P. K. and Carbonell, R. G., Biotechnology and Bioengineering, 36, 572 (1990).
Biomolecules (e.g., interferons, hormones, immunoglobulins, growth factors, DNAs, etc.) obtained from large-scale fermentation and cell-culture processes may be present in low concentrations in a complex medium/broth containing various combinations of cells, cell fragments, lysed cells, colloidal materials, etc. For example, the medium may be a mixture of an aqueous solution and particles. By way of another example, the medium could be proteins dissolved in water. The nature of such heterogeneous aqueous solutions containing the biomolecules is influenced by the nature of the bioproduct, i.e., whether the product is extracellular or intracellular. In the case of intracellular products, cells are recovered from the broth; then cell lysis and homogenization are undertaken to produce a homogenate. The biomolecules are next separated from the cell debris by lysate clarification. In the case of extracellular products, the biomolecules are separated from the whole cells by clarification.
A number of different technologies or sequence of technologies can be employed to eliminate the cellular and colloidal material prior to bioproduct purification via adsorption/chromatography steps. These include centrifugation, flocculation, liquid-liquid extraction and various forms of microfiltration (dead-end, tangential flow and rotary). The devices involved in these processes are complex; there is significant loss of product at each step. See: van Reis, R., L. C. Leonard, C. C. Hsu and S. E. Builder, Industrial scale harvest of proteins from mammalian cell culture by tangential flow filtration, Biotech. Bioengg., 38, 413 (1991). It would be of great use if a process and an apparatus were there to recover and purify the product biomolecule from the whole broth (for extracellular products) or a homogenate (for intracellular products) in one step.
Toward this one-step approach, three solutions have been suggested which employ specialized adsorbent beads/particles.
Nigam et al. (1988) have suggested using specially prepared immobilized adsorbents consisting of small, porous adsorbent particles entrapped within a reversible hydrogel matrix which excludes colloidal contaminants and suspended solids. See: Nigam, S. C., A. Sakoda and H. Y. Wang, Bioproduct recovery from unclarified broths and homogenates using immobilized adsorbents, Biotech. Prog., 4(3), 166 (1988).
Chang and Chase (1996) have employed "streamline" adsorbents specially designed by Pharmacia Biotech (Uppsala, Sweden) for use in expanded bed adsorption of biomolecules from unclarified feedstocks. See the Chang, Y. K. and H. A. Chase, Ion exchange purification of G6PDH from unclarified yeast cell homogenates using expanded bed adsorption, Biotech. Bioengg., 49, 402 (1996).
Aagesen et al. (1995) have designed beads incorporating dense inert particles so that they have a significantly higher density and can settle easily from an expanded fluidized bed after adsorbing the protein of interest from the solution; this technique has been identified as upfront chromatography (UFC) and the beads are identified as UpFront matrix. See: Aagesen, M., T. Wickborg and A. Lihme, Single-step initial protein purification with UpFront Chromatography, Gen. Eng. News, p-12, Apr. 1, 1995.
All of these aforementioned techniques require specially designed and costly adsorbents and/or unusual operational conditions in expanded/fluidized beds to accommodate the presence of an unclarified broth. More often than not, the specially designed bead may not have the required ion exchange or other ligands for the biomolecule separation from solution. On the other hand, microfiltration-based cell-protein separation or lysate clarification are being increasingly employed in small as well as large-scale harvesting of proteins and other biomolecules. Cf. van Reis et al. (1991), supra. Further, a wide variety of adsorbent beads or chromatographic matrix particles are commercially available and routinely used for biomolecule purification.
An object of the present invention is to efficiently integrate these functions into one device using commercially available and commonly utilized microfiltration membranes and adsorbent beads.
Various bioseparation-type devices have been proposed.
One type of device employs an adsorption bed with a hollow fiber housing, wherein a hollow fiber module was used as a housing for adsorbent beads, as described by Pan and McMinis in their U.S. Pat. No. 5,139,668 (1992), wherein adsorbent beads were "emplaced" on the tube side of the hollow fiber module, or, in another case, on the shell side of the module. The device was intended for gas or liquid separations and thus represented certain advantages over conventional packed bed elements or columns, including: the fluid pressure drop through the element is independent of the size of the particles because the fluid flow path through the fiber bore is separated from the particles in the case of particles on the shell side; very fine particles can, therefore, be used on the shell side; the microporous hollow fibers provide efficient and uniform contact between the adsorbent particles and the fluid mixture for a wide range of flow rates, etc. Thus, the hollow fiber modules provided a better housing for some adsorbent beads for certain applications as compared to the conventional packed bed adsorption columns. Notably, however, the hollow fibers were only used as the housing for adsorbent particles and the fibers themselves did not play any role in the separation. Further, no flow stream was ever taken out through the particle side.
Another approach to bioseparation involved the simultaneous ultrafiltration and affinity sorptive separation of proteins in a hollow fiber membrane module as reported by Molinari, R, J. L. Torres, A. S. Michaels, P. K. Kilpatrick and R. G. Carbonell, in "Simultaneous Ultrafiltration and Affinity Sorptive Separation of Proteins in a Hollow Fiber Membrane Module," Biotechnol. Bioeng., 36, 572 (1990), wherein sorptive gel particles were loaded into the shell side of a hollow fiber membrane module, and the device was used in a process for simultaneous protein ultrafiltration and adsorption. In the process of Molinari et al., long binding times (seven hours in the example of horse serum cholinesterase, and five hours in the example of bovine liver carboxylesterase) were used to load proteins onto the adsorbent particles by recirculating the retentates, while the proteins were always present in the filtrates. At the end of the loading step, breakthrough occurred and the adsorbent bed was completely saturated by the protein. Furthermore, in the process of Molinari et al., the elution was conducted by permeating an eluent through the fiber lumen into the shell space. Since the bed was saturated by the feed protein, the desorption was similar to that in a conventional batch adsorption process. No chromatographic purification or fractionation took place.
However, in the present invention, as discussed herein, the mode of operation of the inventive device/process is appropriate for chromatographic fractionation of proteins through a bed of absorbents. Moreover, the chromatographic bed of the present invention is never saturated during the loading step.
Yet another type of device and process for bioseparation involves moving adsorbent particles through the lumen of membrane filters, wherein adsorbent particles binding the target compound are circulated through the lumen of a tubular microfiltration membrane or the lumen of a hollow fiber membrane module. The separation occurs when the compound bound to the particles is retained together with the particles and the compound not bound to the particles permeates through the membrane. This type of device and process were described in three patents by Byers et al., Canadian Patent 1,292,952 (1991), Degen et al., U.S. Pat. No. 5,567,615 (1996), and Pirbazari and Badriyha, U.S. Pat. No. 5,505,841 (1996) respectively. While the former two patents were aimed at biomolecule separations, the third was directed toward water decontamination. In Byers et al. (1991), for example, adsorbent beads were added to a mixture consisting of the target biomolecule and impurities, and the resulting solution was mixed to allow the adsorption of the target biomolecule onto the beads. The suspension was then circulated through the lumen of hollow fiber membranes. The target biomolecule was retained with the beads due to the large size of the beads. The impurities which were not bound to the beads permeated through the membrane to the shell space.
A principal object of the present invention is to provide a method and device for isolation and purification of biomolecules.
Another object of the present invention is to provide a method and apparatus which integrates clarification, concentration and separation of biomolecules into a single step or single device.
It is another object of the present invention to provide an apparatus and method which may be easily adopted for large scale processing.
Another object is to provide a hybrid bioseparation apparatus and process involving commercially available membranes and adsorbents.
A further object is to provide a bioseparation method and apparatus suitable for both extracellular and intracellular products.
Other objects of the present invention, as well as particular features, elements, and advantages thereof, will be elucidated in, or be apparent from, the following description and the accompanying drawing figures.